This application addresses broad Challenge Area (06) Enabling Technologies and specific Challenge Topic 06-ES-102*: 3-D or virtual models to reduce use of animals in research: Creation of miniature multi-cellular organs for high throughput screening for chemical toxicity testing. Development of novel micro-scale systems of multiple cell types that replicate the macro-scale structure and function of major organ systems in response to environmental stressors linked with development of computational models of organ system function can accelerate testing of the multitude of chemicals in our environment for toxicity. Research which furthers the generation of 3-D biological models will provide new assays for rapid screening of toxicity in organs such as the lung and liver. Cell types, such as human stem cells, used in these systems would reduce the use of animals and improve our assessment of chemical hazards in the environment. Contact: Dr. David Balshaw, balshaw@niehs.nih.gov, (919) 541-2448 Title: Computational and Cell Culture Models of Mucus Clearance. Summary: The lung inhales over 1 million infectious and toxic agents each day. The defense of the lung begins with the layer of mucus that lines the epithelial cell layer. When these assaults land at the mucus/air inter- face, the race between infection and clearance begins. The particulates, pathogens and allergens are in a diffusive race for the cell membrane before they can be cleared by the entraining flow of mucus. To provide effective clearance, the body must accomplish several goals. First it must provide the barrier layer of mucus with a height and viscoelasticity that impose a significant transit time for the infectious agents. Second, propulsion mechanisms through cilia or airflow must move the mucus with a speed that clears the agents before they can diffuse to the cell membrane. Third, the viscoelasticity of the mucus must be sufficient to prevent flooding of the airway, yet be fluid enough to allow for transport by cilia and airflow. The goal of this project is to generate computational and cell culture based models for mucus clearance. Since failure of clearance can be under- stood as the first step to a cascade of lung failure scenarios, the establishment of effective model systems for toxicity testing is critical. The use of a cell culture based model allows the inclusion of complex biochemical and immunological responses. The use of ciliated cultures that have been shown to generate and maintain appropriate mucus layers, and to generate a coordinated array of cilia for propulsion, is the starting point for our model system. We will generate the first ciliated cell culture systems that place the cells into a geometry that replicates essential features of the lung: directional, linear flow with converging cross section that can be challenged for vertical transport against gravity. This model system will have the potential to act as a sensitive as- say for environmental effectors that compromise mucus height regulation, mucus rheology, cilia density and co- ordination. We will further develop the assay within a microfluidic system that has twelve isolated assays operating in parallel. This will bring this sophisticated cell based assay to medium-throughput screening. Beyond biophysical models, we require a computational model so that we may predict the consequences of environmental assaults and design effective therapies. We will develop theoretical models that in- corporate the propulsion mechanisms of cilia and of airflow. The latter is operative during effective clearance maneuvers such as cough, and it is understood that even in the absence of cilia propulsion, cough can maintain sterile airways. However, there is currently no predictive model of the role of mucus rheology and layer thickness, cilia effectiveness and airflow in producing sufficient clearance to maintain healthy lung function. By combining a team of researchers from Applied Mathematics, Physics, Biochemistry and Biophysics, and the UNC Cystic Fibrosis Center, the goal of this project is to develop cell-based biophysical assays that can test environmental assaults for their role in compromising mucus clearance, and use them to establish a computational model for clearance that will have the potential for creating in-silico testing of toxins. The goal of this targeted two year project is to jump-start technical advances, in cell cultures and mathematical modeling, that will contribute to the vision of effective, efficient and physiologically accurate toxicity assays. The availability of a cell culture-based clearance assay coupled with an in- silico computational model will reduce the reliance on animal models and more accurately predict the consequences of toxins on human health.